The maintenance of pluripotence is central to the function of embryonic stem (ES) cells, without which ES cells will spontaneously differentiate. Significant headway has been made in understanding the maintenance of pluripotence in mouse ES (mES) cells; however, little is known about the maintenance of pluripotence in human ES (hES) cells, which is crucial for their practical application in directed differentiation for tissue engineering. In order to maintain hES cells in long-term culture and maintain pluripotence, as is possible with mES cells, we need to understand the factors that govern the maintenance of pluripotence in hES cells. A good starting point is to determine whether the information that has been learned in mES cells is applicable to hES cells. The transcription factors Oct4 and Sox 2, which regulate FGF4 expression, have been shown in the mouse to be essential for the maintenance of ES cell function. The factors that positively and negatively regulate the expression of Oct4 and Sox2 in hES cells also play essential roles in regulating pluripotence. My laboratory has focused on the regulation of the Oct 4 gene, which is regulated by several members of the nuclear receptor family including retinoid receptors and SF-1, which regulate positive expression of Oct4, and negative factors such as the orphan receptor GCNF. In mice we have shown that GCNF is essential for repressing Oct4 expression in somatic cells during gastrulation and thus inversely correlates with pluripotence. We propose to determine if the same regulation of Oct4 exists in hES cells by carrying out the following specific aims: First we will determine the stability of expression of Oct4 in hES cells and determine if there is loss of Oct4 expression in any of the cells. Then determine whether the loss of Oct4 expression correlates with loss of SF-1 or gain of GCNF expression. Then we will perform differentiation studies to determine if there is a reciprocal expression pattern for Oct4 and SF-1 in undifferentiated cells and GCNF in differentiated cells. Lastly we will perform RNAi to knock down the expression of GCNF, and SF-1 and look at the effect on Oct4 expression to determine if SF-1 is required to maintain Oct4 expression and whether GCNF is required for Oct4 repression. The culmination of these specific aims will shed new light on some of the factors that regulate pluripotence in hES cells.